Genes for enhancing resistance to Magnaporthe oryzae and uses thereof

ABSTRACT

The present invention relates to Pi5-1 and Pi5-2 proteins which enhance resistance to  Mag - naporthe oryzae , genes which encode the proteins, a recombinant vector comprising the genes, a plant transformed with the recombinant vector and seeds thereof, a method of increasing resistance to a plant pathogen by expressing the genes in a plant, antibodies against the proteins, and a composition comprising the genes which are useful for enhancing resistance to a plant pathogen.

TECHNICAL FIELD

The present invention relates to genes for enhancing resistance to Magnaporthe oryzae and use thereof. More specifically, the present invention relates to Pi5-1 and Pi5-2 proteins which enhance resistance to Magnaporthe oryzae, genes which encode the proteins, a recombinant vector comprising the genes, a plant transformed with the recombinant vector and seeds thereof, a method of increasing resistance to a plant pathogen by expressing the genes in a plant, antibodies against the proteins, and a composition comprising the genes which are useful for enhancing resistance to a plant pathogen.

BACKGROUND ART

The innate immune response is critical to the survival of plants and animals. The response is mediated by the detection of pathogen-associated molecular patterns (PAMPs) (also referred to as microbe-associated molecular patterns) or avirulence (Avr) proteins by pathogen recognition receptors (PRRs; also called pattern recognition receptors or disease resistance proteins). In animals, a family of cytosolic PRRs that contain a nucleotide-binding oligomerization domain (NOD) mediates the apoptotic and inflammatory responses critical to protection from pathogen invasion. Plants also contain a set of intracellular PRR proteins, called NB-LRR (nucleotide binding-leucine rich repeat) R proteins, which are structurally similar to animal NOD proteins. These plant NB-LRR proteins are characterized by a tripartite domain architecture consisting of an N-terminal coiled-coil (CC) or Toll/interleukin-1 receptor (TIR) domain, a central NB domain, and a C-terminal LRR domain (Hammond-Kosack and Jones (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 575-607) and typically recognize pathogen-derived Avr proteins (also called effectors) (Van der Biezen and Jones (1998) Trends Biochem. Sci. 23: 454-456).

Rice blast is one of the most devastating diseases of rice and occurs in all areas of the world where rice is cultivated. More than 70 blast R genes that confer resistance to geographically different sets of the rice blast pathogen Magnaporthe oryzae isolates have been identified to date (Ballini et al. (2008) Mol. Plant. Microbe Interact. 21: 859-868). For example, Pib confers robust resistance to a majority of the Japanese Magnaporthe oryzae isolates (Wang et al. (1999) Plant J. 19: 55-64). In contrast, Pi37 confers only partial resistance to Japanese isolates but complete resistance to Chinese isolates of the same pathogen. Hence, the isolation of multiple R genes is required to fully understand the molecular basis of the resistance to rice blast. Such characterization of these genes will facilitate development of agronomically useful rice cultivars through markerassisted breeding or through transgenic approaches.

To date, a total of nine rice blast resistance genes have been cloned and characterized: Pib, Pita, Pi9, Pi2 and Piz-t, Pi-d2, Pi36, Pi37, and Pikm. With the exception of Pi-d2, a non-RD receptor-like kinase, these genes all encode NB-LRR type proteins. Distinct features of these cloned rice blast resistance genes have been observed. The Pib protein contains a duplicated NB region. Pita lacks a classic LRR but contains a leucine-rich domain (LRD) consisting of imperfect repeats of various lengths. A single amino acid difference at the Pita LRD was found to distinguish resistant from susceptible alleles. The allelic genes Pi2 and Piz-t show 8 amino acid differences within three consecutive LRRs, and these residues are responsible for resistance specificity. The Pi9 gene strongly resembles the Pi2 and Piz-t genes and is located within the same region on chromosome 6. The Pikm-mediated resistance requires two adjacent NB-LRR genes Pikm1-TS and Pikm2-TS. Among these cloned R genes, only Pita has been observed to interact with the corresponding Magnaporthe oryzae avirulence protein, AvrPita. Thus, defense signaling mediated by NB-LRR type proteins remains poorly characterized in rice.

It has been reported that Pi5 confers resistance to many Magnaporthe oryzae isolates collected from Korea and the Philippines (Wang et al. (1994) Genetics 136: 1421-1434). To gain a further understanding of the molecular basis of Pi5-mediated rice blast resistance, we used a map-based method to isolate the Pi5 genomic region. We previously mapped Pi5 to a 170-kb interval on the short arm of chromosome 9 in the RIL260 rice cultivar (Jeon et al. (2003) Molecular Genetics and Genomics 269: 280-289).

According to Korean Patent Registration No. 10-0764563, a gene for inducing resistance to a plant disease, a vector comprising the gene and a transformant obtained from the vector are described. Furthermore, according to Korean Patent Registration No. 10-0701302, plant disease-resistant ogpr 1 gene separated from wild rice, the amino acid sequence of the gene, and a transformant using the same are described. However, said gene is different from the gene of the present invention.

DISCLOSURE OF INVENTION Technical Problem

The present invention is devised in view of the above-described needs. Specifically, in order to further narrow Pi5 resistant gene locus based on a new mapping population, two types of CC-NB-LRR genes, which are candidate genes for Pi5, were identified by sequence analysis of the genomic region having resistance. In addition, transgenic rice plants expressing one or both of said candidate genes were produced and their phenotypes resistant to Magnaporthe oryzae were characterized.

Technical Solution

In order to solve the problems described above, the present invention provides Pi5-1 and Pi5-2 proteins which enhance resistance to Magnaporthe oryzae.

Further, the present invention provides genes which encode the proteins.

Further, the present invention provides a recombinant vector comprising the genes.

Further, the present invention provides a plant transformed with the recombinant vector and seeds thereof.

Further, the present invention provides a method of increasing resistance to a plant pathogen by expressing the genes in a plant.

Further, the present invention provides antibodies against the proteins.

Still further, the present invention provides a composition comprising the genes which are useful for enhancing resistance to a plant pathogen.

Advantageous Effects

According to the present invention, based on coordination between the Pi5-1 gene and the Pi5-2 gene, resistance to a plant pathogen, in particular Magnaporthe oryzae, can be enhanced.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represents chromosomal location of the Pi5 locus in the RIL260/IR50 population. (top) The 170-kb Pi5 resistance genomic region is shown between the markers C1454 and S04G03 in RIL260/CO39 and RIL260/M202. (bottom) A schematic diagram of the 8 rare recombinants in the Pi5 region identified in the RIL260/IR50 population. Breakage points are indicated between the relevant molecular markers. Open bars indicate the presumed RIL260 genome, black bars indicate the IR50 genome, and shaded bars indicate that the region is heterozygous between the two genomes. The bold arrow indicates the 130-kb minimal interval carrying the Pi5 locus, delimited by analysis of the mapping population. Resistance to Magnaporthe oryzae PO6-6 were determined in the F₃ progeny of each line. R, resistant; S, susceptible; R/S, segregating line.

FIG. 2 represents genomic sequence comparison at the Pi5 loci in the RIL260 and Nipponbare cultivars. Predicted ORFs determined by RiceGAAS are shown for both genomes. NB-LRR genes, Pi5-1 alleles, and the Pi5-2 and Pi5-3 genes are indicated by black arrows. The N-terminal region of the Pi5-1Nipponbare allele that is absent in RIL260 is shown in green. Putative transposons and hypothetical genes are indicated by blue and gray arrows, respectively. Open arrows with numbers are predicted to encode the following proteins: 1, putative eukaryotic translation initiation factor; 2, putative GTP-binding protein; 3, putative tetrahydrofolate synthase; 4, putative aldose 1-epimerase; 5, putative histone H5; 6, putative cold-shock DEAD-box protein A; 7 and 10, ankyrin-like proteins; 8 and 9, HGWP-repeat containing proteins. The red dashed line indicates high similarity (>90%) between the RIL260 and Nipponbare ORFs. The chromosomal region that shows little or no homology is indicated by a thin line. The arrows indicate the direction of transcription. A gap in the DNA sequence in RIL260 is indicated by the dotted box.

FIG. 3 represents analysis of transgenic rice plants. (A) RT-PCR analysis of Pi5-1, Pi5-2 and Pi5-1/Pi5-2 F₁ transgenic rice plants 2 days after inoculation with Magnaporthe oryzae PO6-6. The rice Actin1 gene was used as an internal control in these reactions. (B) Disease symptoms in Pi5-1, Pi5-2, and Pi5-1/Pi5-2 F₁ transgenic plants 7 days after inoculation with Magnaporthe oryzae PO6-6. (C) Genomic DNA PCR analysis and disease reaction of F₂ progeny derived from Pi5-1-63/Pi5-2-74 F₁ transgenic plants in response to Magnaporthe oryzae PO6-6 infection. A resistant cultivar, RIL260 carrying Pi5, and a susceptible cultivar, Dongjin (DJ) lacking the Pi5 gene, were used as controls.

FIG. 4 represents genomic structure of the Pi5-1 and Pi5-2 genes and their gene products. Exons are indicated by light gray boxes and introns are indicated by bold lines. 5′- and 3′-untranslated regions (UTR) are indicated by dark gray boxes. ATG and TGA denote the translation initiation and stop codons, respectively and the numbers indicate the amino acid positions.

FIGS. 5 and 6 represent amino acid sequences of Pi5-1 protein (SEQ ID NO: 1) and Pi5-2 protein(SEQ ID NO: 2), respectively. Both resistance proteins contain a coiled-coil (CC), nucleotide-binding site (NB), leucine-rich repeat (LRR), and C-terminal region (CT). Amino acids 31-67 of Pi5-1 SEQ ID NO: 1) and 26-87 of Pi5-2 (SEQ ID NO: 2), shown in underlined italics, contain CC motifs. The conserved internal motifs characteristic of NB proteins, namely the P-loop, kinase-2, RNBS-B, GLPL, RNBS-D, and MHDV domains, are underlined and in bold. A conserved xLDL motif found in the LRR of many NB-LRR proteins is also underlined.

FIG. 7 represents RT-PCR analysis of the Pi5 genes and the PBZ1 gene in the RIL260 cultivar inoculated with Magnaporthe oryzae PO6-6. cDNAs prepared from the leaf tissue of RIL260 at 0, 4, 12, 24, 48 and 72 hr after pathogen inoculation were used in the experiment. The rice Actin1 gene was used as an internal control.

MODE FOR THE INVENTION

In order to achieve the purpose of the invention as described above, the present invention provides Pi5-1 and Pi5-2 proteins which can enhance resistance to Magnaporthe oryzae. In this regard, each of the Pi5-1 and Pi5-2 proteins cannot independently enhance resistance to Magnaporthe oryzae. Instead, only with coordination between the Pi5-1 and Pi5-2 proteins, enhancement of resistance to Magnaporthe oryzae can be obtained.

The scope of the Pi5-1 and Pi5-2 proteins of the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 that is isolated from rice, and functional equivalents of said proteins. The term “functional equivalent” means that, as a result of addition, substitution or deletion of amino acid residues, it has an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% homology with the amino acid sequence that is represented by SEQ ID NO: 1 or SEQ ID NO: 2, thus indicating a protein which has substantially the same physiological activity as the protein expressed by SEQ ID NO: 1 or SEQ ID NO: 2. The term “substantially the same physiological activity” indicates an activity of enhancing resistance of a plant to Magnaporthe oryzae.

The present invention further provides genes which encode said Pi5-1 and Pi5-2 proteins. Each of the Pi5-1 and Pi5-2 genes of the present invention may also comprise a coiled-coil (CC), a nucleotide-binding (NB) domain and a leucine-rich repeat (LRR) domain (see FIG. 5 and FIG. 6). The genes of the present invention include both genomic DNA and cDNA which encode Pi5-1 and Pi5-2 proteins. Specifically, cDNA sequence of Pi5-1 includes 5′ and 3′ non-translated regions, each comprising 70 by and 220 bp, respectively, and encodes 1,025 amino acids. cDNA sequence of Pi5-2 includes 5′ and 3′ non-translated regions, each comprising 73 by and 164 bp, respectively, and encodes 1,063 amino acids.

Preferably, each of the genomic DNAs of Pi5-1 and Pi5-2 of the present invention may comprise a nucleotide sequence that is described either by SEQ ID NO: 3 or by SEQ ID NO: 4. Further, each of the cDNAs of Pi5-1 and Pi5-2 of the present invention may comprise a nucleotide sequence that is described either by SEQ ID NO: 5 or by SEQ ID NO: 6. Variants of said nucleotide sequence are also within the scope of the present invention. Specifically, said gene may comprise a nucleotide sequence with at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% homology with the nucleotide sequences of SEQ ID NO: 3 to SEQ ID NO: 6. The “sequence homology %” for a certain polynucleotide is determined by comparing two nucleotide sequences that are optimally arranged with a region to be compared. In this regard, a part of the polynucleotide sequence in a region to be compared may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any addition or deletion) relative to the optimized arrangement of the two sequences.

Further, the present invention provides a recombinant vector comprising the Pi5-1 and Pi5-2 of the present invention.

The term “recombinant” indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene fragment in a form of a sense or antisense, that are not found in natural state of cell. In addition, a recombinant cell can express a gene that is found in natural state, provided that said gene is modified and re-introduced into the cell by an artificial means.

The term “vector” is used herein to refer DNA fragment (s) and nucleotide molecules that are delivered to a cell. Vector can replicate DNA and be independently reproduced in a host cell. The terms “delivery system” and “vector” are often interchangeably used. The term “expression vector” means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequences that are essential for the expression of the operatively-linked coding sequence in a specific host organism.

Preferably, the recombinant vector of the present invention is a recombinant plant expression vector.

A preferred example of plant expression vector is Ti-plasmid vector which can transfer a part of itself, i.e., so-called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vector (see EP 0 116 718 B 1) are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting hybrid DNA to a plant genome. Especially preferred form of Ti-plasmid vector is a so-called binary vector which has been disclosed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other vector that can be used for introducing the DNA of the present invention to a host plant can be selected from a double-stranded plant virus (e.g., CaMV), a single-stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector. Use of said vector can be advantageous especially when a plant host cannot be appropriately transformed.

Expression vector would comprise at least one selective marker. Said selective marker is a nucleotide sequence having a property which allows a selection based on a common chemical method. Any kind of gene that can be used for the differentiation of transformed cells from non-transformed cell can be a selective marker. Example includes, a gene resistant to herbicide such as glyphosate and phosphintricin, and a gene resistant to antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.

For the plant expression vector according to one embodiment of the present invention, a promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but not limited thereto. The term “promoter” means a DNA molecule to which RNA polymerase binds in order to initiate its transcription and it corresponds to a DNA region upstream of a structural gene. The term “plant promoter” indicates a promoter which can initiate transcription in a plant cell. The term “constitutive promoter” indicates a promoter which is active in most of environmental conditions and development states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing a constitutive promoter is not limited in the present invention.

For the above-described terminator, any conventional terminator can be used for the present invention. Example includes, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto. Regarding the necessity of terminator, it is generally known that such region can increase a reliability and an efficiency of transcription in plant cells. Therefore, the use of terminator is highly preferable in view of the contexts of the present invention.

Further, the present invention provides a plant that is transformed with the recombinant vector according to the present invention.

The plant according to the present invention is preferably a monocot plant including rice, barley, maize, wheat, rye, oat, turf grass, hay, millet, sugar cane, rye grass, orchard grass, and the like. Most preferably, it is rice.

Further, the present invention provides seeds of the above-described plants. Preferably, the seeds are rice seeds.

Further, the present invention provides a method of increasing resistance to a plant pathogen, comprising steps of transforming a plant with the recombinant vector of the present invention which includes the Pi5-1 and Pi5-2 genes and then expressing the Pi5-1 and Pi5-2 genes in the plant. Preferably, the pathogen is Magnaporthe oryzae. The above-described plant is preferably a monocot plant including rice, barley, maize, wheat, rye, oat, turf grass, hay, millet, sugar cane, rye grass, orchard grass, and the like. Most preferably, it is rice.

Plant transformation means any method by which DNA is delivered to a plant. Such transformation method does not necessarily have a period for regeneration and/or tissue culture. Transformation of plant species is now quite general not only for dicot plants but also for monocot plants. In principle, any transformation method can be used for introducing a hybrid DNA of the present invention to an appropriate progenitor cells. It can be appropriately selected from a calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), an electroporation method for protoplasts (Shillito R. D. et al., 1985 Bio/Technol. 3, 1099-1102), a microscopic injection method for plant components (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), a particle bombardment method for various plants components (DNA or RNA-coated) (Klein T. M. et al., 1987, Nature 327, 70), or a (non-complete) viral infection method in Agrobacterium tumefaciens mediated gene transfer by plant invasion or transformation of fully ripened pollen or microspore (EP 0 301 316), etc. A method preferred in the present invention includes Agrobacterium mediated DNA transfer. In particular, so-called binary vector technique as disclosed in EP A 120 516 and U.S. Pat. No. 4,940,838 can be preferably adopted for the present invention.

The term “plant cell” that is used for the plant transformation according to the present invention can be any plant cell. The plant cell can be a cultured cell, a cultured tissue, a cultured organ, or a whole plant, preferably a cultured cell, a cultured tissue or a cultured organ, and more preferably any form of a cultured cell. Preferably, the plant is rice.

The term “plant tissue” includes either differentiated or undifferentiated plant tissue, including root, stem, leaf, pollen, seed, cancerous tissue and cells having various shape that are used for culture, i.e., single cell, protoplast, bud and callus tissue, but not limited thereto. Plant tissue can be in planta or in a state of organ culture, tissue culture or cell culture.

Further, the present invention provides antibodies against the Pi5-1 and Pi5-2 proteins of the present invention. According to the present invention, the term “antibody” includes a monoclonal antibody, a multi-specific antibody, a human antibody, a humanized antibody, a camelised antibody, a chimera antibody, a single-chain Fvs (scFv), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab) fragment, a disulfide-bonding Fvs (sdFv), an anti-idiotype (anti-Id) antibody, or a fragment which binds to any of the above-described epitopes. In particular, an immunoglobulin molecule and an immunologically-active fragment of an immunoglobulin molecule, i.e., a molecule which comprises an antigen-binding region, are included in the antibody of the present invention. An immunoglobulin molecule can be any kind including IgG, IgE, IgM, IgD, IgA, and IgY or any class including IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂ or their subclass.

The antibody of the present invention can be prepared according to a general method which comprises steps of cloning the genes of the present invention in an expression vector by following a typical procedure to obtain a protein and preparing the antibody from the protein. Herein, a partial peptide which can be generated from the protein is also included. As for a partial peptide of the present invention, it comprises at least seven amino acids, preferably at least nine amino acids, and more preferably at least twelve amino acids. Type of the antibody of the present invention is not specifically limited. A monoclonal antibody, a polyclonal antibody and a part of such antibodies having an antigen-binding property are all included in the antibody of the present invention. All kinds of immunoglobulin antibody are also included. Furthermore, a special antibody such as a humanized antibody and the like is also included in the antibody of the present invention.

Still further, the present invention provides a composition comprising the Pi5-1 and Pi5-2 genes for enhancing resistance to a plant pathogen. Since the Pi5-1 and Pi5-2 proteins of the present invention enhance resistance to a plant pathogen based on coordination between them, the composition comprising the Pi5-1 and Pi5-2 genes can be used for enhancing resistance to a plant pathogen. Preferably, the plant pathogen is Magnaporthe oryzae.

The present invention will now be described in greater detail with reference to the following examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples.

EXAMPLES Plant Materials

The RIL260 rice cultivar carrying the Pi5 allele and a rice blast-susceptible cultivar, IR50, were used as the parental lines in this study. The RIL260 and IR50 cultivars were crossed to generate a mapping population for genetic linkage analysis. Self-pollinated seeds (F₂) of the RIL260/IR50 F₁ individuals were collected to obtain a sufficiently large mapping population. A japonica rice cultivar, Dongjin, was used as the susceptible control in the Magnaporthe oryzae inoculation and rice transformation experiments. RIL260 and the monogenic rice line IRBL5-M carrying Pi5 were used as the resistant control cultivars in the Magnaporthe oryzae inoculation experiments. An additional 8 monogenic rice lines, IRBL1-F5, IRBL9-W, IRBLb-B, IRBLta-K1, IRBLz-Fu, IRBLks-F5, IRBLkm-Ts, and IRBLsh-S, and the susceptible background cultivar of these monogenic lines, Lijiangxintuanheigu (LTH) were also used in the inoculation experiments to determine the virulence pattern of Magnaporthe oryzae isolates. Rice seedlings were grown in a greenhouse at 30° C. during the day and at 20° C. at night in a light/dark cycle of 14/10 hr.

Pathogen Inoculation and Disease Evaluation

Magnaporthe oryzae PO6-6, a Philippine isolate, which is incompatible with the Pi5 resistance locus, has been commonly used to detect this locus. To analyze blast resistance in Pi5 transgenic rice plants, an additional 5 different Korean Magnaporthe oryzae isolates, KJ105a, KJ107, KJ401, KI215, and R01-1, were used. All inoculations and disease evaluations were conducted in the greenhouse facilities at Kyung Hee University using a method that was slightly modified from Liu et al. (2002, Mol. Genet. Genomics 267: 472-480). Three week-old plants of the F₃ progeny of each of the identified recombinant lines and transgenic plants were used in the inoculation experiments. Magnaporthe oryzae was grown on oatmeal agar medium for 2 weeks at 24° C. in the dark. Conidia were induced 4 days prior to collection by scratching the plate surface with a sterilized loop. The inoculated plants were placed in sealed containers to maintain humidity at 24° C. in darkness for 24 hr, and then transferred to a growth chamber at 24° C. and 80% humidity under a 14/10-hr (light/dark) photoperiod. Disease evaluation was carried out 7 days after inoculation.

Genotypic Analysis of Progeny from the RIL260/IR50 Mapping Population

Cleaved amplified polymorphic sequence (CAPS) markers for C1454 and JJ817 and a sequence characterized amplified region (SCAR) marker JJ803 (corresponding to the previously reported dominant marker JJ80-T3) were used for the analysis of the RIL260/IR50 segregating progeny (Table 1). The dominant markers JJ113-T3 and S04G03 were additionally utilized as needed.

TABLE 1 PCR primers used in this study Marker Forward primer Reverse primer or gene (5′-3′) (5′-3′) C1454 GTATTACCTGAAATCCTA AGGAACTACGGTATTACA GTGGTG AGGATC (SEQ ID NO: 7) (SEQ ID NO: 8) JJ817 GATATGGTTGAAAAGCTA ATCATTGTCCTTCATATT ATCTCA CAGAGT (SEQ ID NO: 9) (SEQ ID NO: 10) JJ803 AAGTGAGCATCCAGTGCC AGCCGGTGCTCATAACAC TAATGA GTATTA (SEQ ID NO: 11) (SEQ ID NO: 12) Pi5-1 TACAAGTTGGCAGCTTTA TCAGAAGCACTGGATCTT TCTGAG TCTGCA (SEQ ID NO: 13) (SEQ ID NO: 14) Pi5-2 AGTGAACTCCAAACATGT TCATACCTGTTGCGGTTT GAACAC CTGCCT (SEQ ID NO: 15) (SEQ ID NO: 16) Actin1 GGAACTGGATAGGTCAAG AGTCTCATGGATACCCGC GC AG (SEQ ID NO: 17) (SEQ ID NO: 18) PBZ1 ACCATCTACACCATGAAG GTATTCCTCTTCATCTTA CTTAAC GGCGTA (SEQ ID NO: 19) (SEQ ID NO: 20)

Genomic DNA was isolated from young leaves of rice plants using a simple miniprep method (Chen and Ronald (1999) Plant Mol. Biol. Rep. 17: 53-57). PCR analysis was performed in a final volume of 30 μl (100 pM of each primer, 200 μM each of dNTPs, 10 mM Tris-HCl pH 9.0, 2 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100 and 0.5 U Taq polymerase) using 50 ng of genomic DNA as template. PCR products for the CAPS markers C1454 and JJ817 were subsequently digested with Mlu I and AseI, respectively, and then size-fractionated on agarose gels.

DNA Sequencing and Gene Prediction

RIL260 Binary BAC (BIBAC) clones spanning the Pi5 locus were selected for DNA sequencing analysis. Plasmids purified by a minipreparation were partially digested with Sau3AI and separated by agarose gel electrophoresis. The 0.5-3.0 kb genomic DNA fragments were isolated using a commercial kit (Gel Extraction Kit, Qiagen), subcloned into the BamHI site of pBluescriptII SK(−) (Clontech), and then transformed into E. coli DH10B by electroporation. For DNA sequencing of each BIBAC clone with a 25-kb average insert size, about 60 clones were selected and sequenced in one or both directions using the T3 and T7 primers.

Similarity searches against the NCBI database (http://www.ncbi.nlm.nih.gov/) were performed using BLAST (Basic Local Alignment Search Tool). To predict proteincoding gene regions, the Rice Genome Automated Annotation System (RiceGAAS) was utilized (http://RiceGAAS.dna.affrc.go.jp/).

Vector Construction for Genetic Complementation Experiments

Genomic DNA regions for Pi5-1 and Pi5-2 were reconstituted by subcloning from BIBAC clones (Jeon et al. (2003) Mol. Genet. Genomics 269: 280-289). To construct a clone carrying the entire Pi5-1 coding region, a 6.6-kb BamHI-SacI fragment of the JJ80 vector that includes the 0.5-kb predicted promoter was subcloned into the binary vector pC1300intC (GenBank accession no. AF294978). The resulting plasmid JJ104 was digested with BamHI and BstEII and fused to 7.3-kb HindIII-BstEII insert of JJ106 to construct JJ105 with a 5.2-kb promoter region. The 0.5-kb SacI-XhoI fragment was amplified by PCR using primers 5′-GTCCAAAGAGAAATGCGACAACAC-3′ (SEQ ID NO: 21) and 5′-CGCTCGAGGTGGCATTTCATCCAATAGGCAAC-3′ (SEQ ID NO: 22). The resulting product was inserted into the JJ105 to extend the terminator region, yielding the JJ204 construct carrying the 11,516-bp Pi5-1 genomic region.

The Pi5-2 gene was constructed by the multiple ligation of the following four fragments: a 4.2-kb EcoRI-BglII DNA fragment of JJ113, a 200-bp BglII-ClaI PCR product amplified using the primers 5′-GGATGATGTGATCTGCAGAGAAAC-3′ (SEQ ID NO: 23) and 5′-CAGCCTCACTGAAATTGCGAAGCA-3′ (SEQ ID NO: 24), a 4.2-kb ClaI-XbaI DNA fragment of JJ120, and an EcoRI-XbaI digested pC1300intC vector fragment. In the resulting construct JJ117, the promoter region was extended by cloning the 3.7-kb NsiI-EcoRI fragment of JJ120. Finally, by inserting a 0.9 kb-extended terminator sequence into the Eco065I site of the JJ142 plasmid, the 13,250-bp entire genomic sequence of Pi5-2 in JJ212 was constructed. The cloned genomic sequences in JJ204 and JJ212 were confirmed by DNA sequencing.

Production of Transgenic Rice Plants

Genomic clones for Pi5-1 and Pi5-2 were transformed into Agrobacterium tumefaciens EHA105 or LBA4404 by electroporation and introduced into the susceptible rice cultivar Dongjin via Agrobacterium mediation according to an established procedure (Jeon et al. (2000) Plant J. 22: 561-570). The transgenic plants (T₀) were self-pollinated and T₁ seeds were collected. Homozygous Pi5-1 (Pi5-1-63) and Pi5-2 (Pi5-2-74) transgenic lines were then selected from T₂ progeny resulting from selfpollination of the T₁ lines based on the segregation patterns of the transgenes. F₁ plants carrying both Pi5-1 and Pi5-2 were produced from a cross between Pi5-1-63 and Pi5-2-74 lines, and self-pollinated to produce F₂ plants.

Isolation of Pi5-1 and Pi5-2 cDNAs

Two preparations of total RNA were prepared from rice leaves collected at 24 and 48 hr after inoculation with Magnaporthe oryzae PO6-6 using Trizol reagent (Invitrogen). Purified mRNAs were obtained using the PolyATtract mRNA isolation system (Promega) from each set of total RNA and mixed in a 1:1 ratio for cDNA synthesis. cDNAs larger than 0.5 kb were selected by size fractionation via gel filtration, and a cDNA library was constructed with the Uni-ZAP XR vector (Stratagene). This library was then screened via colony blot hybridizations using probes corresponding to the Pi5-1 and Pi5-2 coding regions, a 570-bp HindIII-KpnI fragment of JJ204 and a 589-bp EcoRV-SpeI fragment of JJ212, respectively. Isolated cDNA clones were analyzed by DNA sequencing.

RT-PCR Analysis

To examine the changes in transcript accumulation in response to pathogen treatment, leaves from each of 10 RIL260, IRBL5-M, and transgenic rice plants inoculated with Magnaporthe oryzae PO6-6 were collected at different time periods for RT-PCR analysis. Total RNA was prepared using Trizol reagent and reversetranscribed with an oligo-dT primer and a First Strand cDNA Synthesis Kit (Roche). First-strand cDNA was used in PCR reactions with gene-specific primers. Primers for the rice Actin1 gene and the pathogenesis-related probenazole-inducible (PBZ1) gene were used as internal controls (Table 1). PCR conditions were as follows: 94° C. for 5 min followed by 28-35 cycles of 94° C., 1 min; 56° C., 1 min; and 72° C., 1 min, and a final extension at 72° C. for 5 min. Three independent amplifications were performed for each primer set.

Example 1 Genetic Characterization of a 130-kb Chromosomal Region Carrying Pi5

Previously, the Pi5 resistance gene was delimited to a 170-kb interval between the two flanking markers S04G03 and C1454 on rice chromosome 9. This finding was the result of our previous analysis of two populations generated by crosses between RIL260 carrying Pi5 and a susceptible cultivar CO39, and between RIL260 and another susceptible cultivar M202 (Jeon et al. (2003) Mol. Genet. Genomics 269: 280-289). To further delineate the Pi5 gene, in the present study, we generated a third mapping population derived from a cross between RIL260 and another susceptible cultivar IR50. Through PCR screening we found that among the susceptible cultivars tested, only IR50 contained the dominant marker JJ817, which was also found in the resistant cultivar RIL260 (data not shown). In contrast, we were not able to amplify a PCR product for JJ817 in other susceptible cultivars including CO39 and M202. We selected IR50 as a mapping parent based on the similarity between the genomic regions for RIL260 and IR50 which we speculated could facilitate recombination in the interval.

To identify rare recombinants within the 170-kb Pi5 locus, a prescreening strategy using the CAPS markers JJ817 and C1454 and a SCAR marker JJ803 was employed in our current analysis of the RIL260/IR50F₂ population. Of the 2,014 F₂ individuals analyzed, we identified 8 recombinants between JJ817 and JJ803, but none between JJ803 and C1454 (FIG. 1). Using the dominant markers JJ113-T3 and S04G03, we subsequently determined the breakage points of the 8 recombinants we isolated in their progeny (F₃) plants, which enabled us to distinguish homozygous from heterozygous genotypes. In total, all 8 lines were found to harbor recombination events between JJ113-T3 and JJ817.

The disease phenotypes resulting from Magnaporthe oryzae PO6-6 infection of these 8 identified lines were then determined in the F₃ progeny in each case. These experiments further delimited the Pi5 gene to a 130-kb interval between the markers JJ817 and C1454 (FIG. 1). Our previous and current results indicated that both the JJ803 and JJ113-T3 markers cosegregate with Pi5-mediated resistance (FIG. 1). We were unable to further fine-map the R gene at the Pi5 locus.

Example 2 Genomic Sequence Analysis of the 130-kb Chromosomal Region Containing the Pi5 Locus

To identify candidate R genes in the Pi5 locus, 7 BIBAC clones, JJ80, JJ98, JJ106, JJ110, JJ113, JJ120, and JJ123, which covered the 130-kb Pi5 region were selected and sequenced. BLAST searches using these sequences against the public databases and also gene annotation analysis using the RiceGAAS program predicted a total of 18 open reading frames (ORFs) at the Pi5 locus in the RIL260 cultivar: 7 hypothetical proteins, 2 NB-LRR proteins, 2 putative transposon proteins, a putative eukaryotic translation initiation factor, a putative GTP-binding protein, a putative tetrahydrofolate synthase, a putative aldose 1-epimerase, a putative histone H5, a putative cold-shock DEAD-box protein A, and an ankyrin-like protein (FIG. 2). From this genomic sequence analysis, two Pi5 candidate genes that showed homology with NB-LRR resistance genes were identified in RIL260 and designated Pi5-1 and Pi5-2.

The region of approximately 90 kb, from JJ803 to JJ817, of the 130-kb RIL260 Pi5 interval was compared with the corresponding region of the japonica genome represented by the sequenced cultivar Nipponbare (International Rice Genome Sequencing Project 2005; FIG. 2). The resulting sequence analysis showed that the NipponbarePi5 interval contains two NB-LRR genes, Os09g15840 (a Pi5-1 allelic gene) and a gene which was not identified in RIL260, Os09g15850, denoted Pi5-3. In contrast, Nipponbare lacks the corresponding allele of Pi5-2. Notably, the 5 upstream sequences of the Pi5-1 allelic genes of RIL260 and Nipponbare were very different, indicating an extreme sequence divergence within the regulatory sequences of these alleles. In addition, we did not observe significant sequence similarity in any other part of the 90-kb Pi5 intervals in RIL260 and Nipponbare (FIG. 2). These results suggest that the Pi5 resistance locus has significantly diverged between these resistant and susceptible rice cultivars.

We did not compare the Pi5 resistance locus with that of the publicly sequenced indica rice cultivar 93-11 due to a large gap at this locus. In an inoculation experiment, we found that both Nipponbare and 93-11 were susceptible to Magnaporthe oryzae PO6-6 (data not shown), indicating neither carries the Pi5 resistance gene.

Example 3 Characterization of Transgenic Rice Plants Expressing Pi5 Candidate Genes

To determine which one of the two candidate genes, Pi5-1 and Pi5-2, is responsible for the Pi5-mediated resistance to M. oryzae, we used the genomic clones JJ204 and JJ212 carrying Pi5-1 and Pi5-2, respectively, under the control of their native promoters to transform the susceptible japonica rice cultivar Dongjin using Agrobacterium-mediated transformation. RT-PCR analysis of the resulting transgenic lines revealed that 13 of 15 Pi5-1 and 12 of 13 Pi5-2 independently transformed lines expressed their transgenes upon Magnaporthe oryzae PO6-6 inoculation (FIG. 3A). The primary transgenic lines (T_(o)) carrying either Pi5-1 or Pi5-2 were inoculated with Magnaporthe oryzae PO6-6. Surprisingly, however, none of the 13 Pi5-1 or the 12 Pi5-2 transgenic plants showed resistance to Magnaporthe oryzae isolate PO6-6 (FIG. 3B). To confirm these results, we inoculated T₁ progeny from these T_(o) lines and found that all progeny were susceptible to the Magnaporthe oryzae isolate to the same extent as the wild-type control Dongjin cultivar. This indicates that neither Pi5-1 nor Pi5-2 alone confers resistance to Magnaporthe oryzae PO6-6.

Example 4 Characterization of Transgenic Rice Plants Expressing Both Pi5-1 and Pi5-2

Because recent reports (Sinapidou et al. (2004) Plant J. 38: 898-909) have demonstrated that the presence of two R genes is required for resistance to pathogen infection, we decided to test plants expressing both candidate genes for blast resistance. We therefore generated transgenic plants carrying both Pi5-1 and Pi5-2 by crossing a highly susceptible homozygous Pi5-I line #63 (Pi5-1-63) with the highly susceptible homozygous Pi5-2 line #74 (Pi5-2-74). Gene expression analysis revealed that the F₁ plants resulting from the cross expressed both the Pi5-1 and Pi5-2 genes upon Magnaporthe oryzae PO6-6 inoculation (FIG. 3A). Strikingly, the 23 of Pi5-1-63/Pi5-2-74 F₁ plants tested all displayed complete resistance to Magnaporthe oryzae PO6-6. Transgenic lines carrying either Pi5-1 or Pi5-2 were susceptible as previously determined (FIG. 3B).

To confirm this finding, we inoculated the F₂ progeny plants from the Pi5-1-63/Pi5-2-74 F₁ lines with the Magnaporthe oryzae isolate PO6-6. Of the 72 F₂ progeny tested, 37 of these carried both transgenes and conferred resistance to Magnaporthe oryzae PO6-6. In contrast F₂ progeny lacking both Pi5-1 and Pi5-2 were susceptible (FIG. 3C). RT-PCR analysis demonstrated that the Pi5-1-63/Pi5-2-74 lines expressed their transgenes at levels that were similar to RIL260 before and after Magnaporthe oryzae PO6-6 inoculation. To test if Pi5-1 and Pi5-2 are required for resistance to other Magnaporthe oryzae isolates, we inoculated the transgenic plants with four additional isolates incompatible with Pi5. These isolates displayed distinct virulence patterns on rice lines carrying different single R genes, validating that these are indeed different Magnaporthe oryzae isolates. We found that transgenic plants coexpressing Pi5-1 and Pi5-2 were resistant to all of the tested Magnaporthe oryzae isolates. The resistance donor RIL260 and the monogenic line IRBL5-M carrying Pi5 were also resistant to these 4 isolates. In contrast, Dongjin and plants carrying either Pi5-1 or Pi5-2 only were susceptible to the tested Magnaporthe oryzae isolates (Table 2). These results demonstrate that the two NB-LRR genes Pi5-1 and Pi5-2 are required for Pi5-mediated resistance to Magnaporthe oryzae isolates.

TABLE 2 Disease reactions of Pi5 transgenic plants to Magnaporthe oryzae isolates IRBL5- Pi5-1-63/ Isolate Dongjin RIL260 M Pi5-1-63^(a) Pi5-2-74^(a) Pi5-2-74^(a) PO6-6 S^(b) R^(b) R S S R KJ105a S R R S S R KJ107 S R R S S R KJ401 S R R S S R R01-1 S R R S S R KI215 S R S S S S ^(a)Transgenic lines. ^(b)R, resistant; S, susceptible.

The Pi5 monogenic line IRBL5-M is susceptible to Magnaporthe oryzae KI215. Genomic sequence analysis indicated that IRBL5-M genomic region carrying Pi5 is identical to that of RIL260 (data not shown). In addition, RT-PCR analysis further demonstrated that IRBL5-M expresses both Pi5-1 and Pi5-2 at levels similar to RIL260 either before or after Magnaporthe oryzae PO6-6 inoculation. Based on these results, we hypothesized that transgenic plants expressing both Pi5-1 and Pi5-2 would also be susceptible to Magnaporthe oryzae KI215. Indeed, our inoculation result showed that transgenic plants expressing both Pi5-1 and Pi5-2 are susceptible to Magnaporthe oryzae KI215. In contrast, RIL260 was found to be resistant to Magnaporthe oryzae KI215, indicating that it may contain an additional R gene that confers resistance to this isolate (Table 2).

Example 5 Characterization and Phylogenetic Analysis of the Proteins Encoded by Pi5-1 and Pi5-2

To isolate the cDNA clones corresponding to both Pi5 genes under study, a cDNA library for RIL260 was constructed with the Uni-ZAP XR vector using mRNA isolated from rice leaves collected at 24 and 48 hr after inoculation with Magnaporthe oryzae PO6-6. This library was screened using a colony hybridization methodology using gene-specific regions of Pi5-1 and Pi5-2 as probes. We identified 7 and 5 cDNA clones for Pi5-1 and Pi5-2, respectively. Sequence analysis further revealed that 3 of the Pi5-1 cDNA clones contained an entire open reading frame (ORF), whereas the others lacked an N-terminus encompassing an ATG translation initiation codon. Among the three full ORF clones, the longest clone (#1-7) was fully sequenced. These experiments revealed that Pi5-1 encodes a protein of 1,025 amino acids and that the ORF is flanked by 5′- and 3′-untranslated regions of 70 and 220 bp, respectively (GenBank accession no. EU869185; FIGS. 4 and 5). Sequence analysis of the Pi5-2 clones revealed that 3 of the 5 clones contained an entire ORF. Among these, the longest clone (#2-4) was further characterized by sequencing. This analysis indicated that Pi5-2 encodes an ORF of 1,063 amino acids and that this ORF is flanked by 5′- and 3′-untranslated regions of 73 and 164 bp, respectively (GenBank accession no. EU869186; FIGS. 4 and 6).

Comparison of their deduced amino acid sequences revealed that both Pi5-1 and Pi5-2 encode an N-terminal CC, a centrally located NB and LRR, and also C-terminal regions (FIGS. 5 and 6). A conserved domain search using the Pfam and SMART databases predicted that residues 109-576 of Pi5-1 and 109-567 of Pi5-2 contain an NB domain, which is a signaling motif shared by plant R gene products. The conserved internal domains characteristic of NB-containing R gene products were also identified in Pi5-1 and Pi5-2, including the P-loop, kinase-2, RNBS-B, GLPL, RNBS-D, and MHDV domains. Additional analysis using the Paircoil2 program (http://groups.csail.mit.edu/cb/paircoil2/) predicted a potential CC domain with a threshold 0.1 between amino acids 31-67 in Pi5-1 and 26-87 in Pi5-2, indicating that these proteins belong to the CC subset of the NB-LRR resistance proteins.

The LRR regions of Pi5-1 and Pi5-2 consist of 24.3 and 22.6% leucine residues, respectively, and contain a series of imperfect repeats (10-12) of various lengths (FIGS. 5 and 6). Of note, a few repeats of the Pi5-1 and Pi5-2 proteins matched the consensus sequence LxxLxxLxxLxLxxC/N/Sx(x) LxxLPxx observed in other cytoplasmic R proteins. The first and third repeat regions of Pi5-1 and the first, third and sixth repeat regions of Pi5-2 contained the xLDL motif that is conserved in the third LRR of many NB-LRR proteins (FIGS. 5 and 6). Notably also, the Pi5-1 and Pi5-2 proteins harbor a unique C terminus that is distinct from those of other NB-LRR proteins, and that does not match any known protein motif.

Sequence comparisons between the cDNA and genomic sequences for these R genes revealed that Pi5-1 and Pi5-2 carry 5 and 6 exons, respectively (FIG. 4). The Pi5 genes have a larger number of introns within their coding regions compared with other cloned rice R genes that confer resistance to M. oryzae. Furthermore, the Pi5-1 and Pi5-2 genes contain an intron in both their RNBS-D and MHDV domains.

Example 6 Expression Analysis of the Pi5-1 and Pi5-2 Genes

To examine whether the expression of the two identified R genes was altered upon pathogen treatment, we performed RT-PCR analysis of these two genes in RIL260, IRBL5-M, and Pi5-1-63/Pi5-2-74 transgenic plants infected with Magnaporthe oryzae PO6-6 (FIG. 7). Total RNAs isolated from the leaves of 3 week-old plants harvested at different time points after Magnaporthe oryzae PO6-6 inoculation were used for this purpose. The results revealed that Pi5-1 expression increased 12 hr after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed at a low level in RIL260 both before and after infection (FIG. 7). The IRBL5-M and Pi5-1-63/Pi5-2-74 lines also exhibited similar expression patterns of the Pi5 genes. These findings indicated that Pi5-1 and Pi5-2 are both expressed during pathogen infection, suggesting that the encoded proteins are also coexpressed. Transcripts of PBZ1, a pathogen-inducible gene, accumulated to high levels in M. oryzae-treated leaves (FIG. 7). 

1. A gene that encodes Pi5-1 protein or Pi5-2 protein, wherein the Pi5-1 protein consists of SEQ ID NO: 1 and the Pi5-2 protein consists of SEQ ID NO: 2, for enhancing resistance to Magnaporthe oryzae.
 2. The gene according to claim 1, wherein genomic DNA of Pi5-1 consists of the nucleotide sequence of SEQ ID NO: 3 and genomic DNA of Pi5-2 consists of the nucleotide sequence of SEQ ID NO: 4 , and cDNA of Pi5-1 consists of the nucleotide sequence of SEQ ID NO: 5 and cDNA of Pi5-2 consists of the nucleotide sequence of SEQ ID NO:
 6. 3. A recombinant vector comprising the gene of claim
 1. 4. A plant transformed with the recombinant vector of claim
 3. 5. The plant according to claim 4, characterized in that it is a monocot plant.
 6. A seed of the plant described in claim
 4. 7. A method of increasing resistance to a plant pathogen, comprising the steps of: transforming a plant with the recombinant vector described in claim 3 and then expressing the Pi5-1 gene or Pi5-2 gene in the plant.
 8. The method according to claim 7, characterized in that the plant pathogen is Magnaporthe oryzae.
 9. The method according to claim 7, characterized in that the plant is a monocot plant.
 10. A composition comprising the gene of claim 1 for enhancing resistance to a plant pathogen. 